Hybridization techniques are well known in the art of molecular biology. They allow for the detection of specific sequences, in case a specific hybridization probe is available Usually, the hybridization probe is labeled with a detectable moiety, e. g. a radioactive or fluorescent label.
In addition, Polymerase Chain Reaction (PCR) has become a powerful and wide-spread technology for analysis of nucleic acids. The principles of PCR are disclosed in U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,102 (Mullis et al.). A major improvement in PCR derives from the possibility of measuring the kinetics of a PCR reaction by On-Line detection. This has become possible by means of detecting the amplicon through fluorescence monitoring.
Alternatively, suitable fluorescently labeled hybridization probes which are already present during the amplification reaction may be used subsequently in a homogenous assay without opening the reaction vessel in order to perform a temperature dependent melting curve analysis. For such an analysis the temperature of the sample is increased continiously and the exact melting temperature is determined at which the previosly generated hybrid complex between (amplified) target nucleic acid and hybridization probe is resolved. Such an approach may be used in order to detect differences in melting temperatures of target molecules which only differ from each other by a single nucleotide polymorphism. In other words, melting curve analysis can even be used for the detection or identification of point mutations.
Examples of such techniques are disclosed in detail in WO 97/46707, WO 97/46712 and WO 97/46714 (Wittwer et al.), the disclosures of which are hereby incorporated by reference.
Several detection formats based on target dependent fluorescent signaling have been disclosed, which enable continuous monitoring of the generation of PCR amplification products or identification of mutations during a subsequent melting curve analysis (reviewed in Wittwer et al., Biotechniques, Vol. 22, No, 1, 130-138, 1997). These detection formats include but are not limited to:
a) Increased Fluorescence Resonance Energy Transfer Upon Hybridization
For this detection format, two oligonucleotide hybridization probes each labeled with a fluorescent moiety are used which are capable of hybridizing to adjacent but non overlapping regions of one strand of the amplification product. Preferably, one oligonucleotide is labeled at the 5xe2x80x2 end and the second oligonucleotide is labeled at the 3xe2x80x2 end. When hybridized to the target DNA, the two fluorescent labels are brought into close contact, such that fluorescence resonance energy transfer between the two fluorescent moieties can take place. As a consequence, the hybridization can be monitored through excitation of the donor moiety and subsequent measurement of fluorescence emission of the second acceptor moiety (WO 97/46714).
In a similar embodiment, only one fluorescently labeled probe is used, which together with one appropriately labeled primer may also serve as a specific FRET pair (Bernard et al., Analytical Biochemistry 235, p. 101-107 (1998)).
b) Molecular beacons
A molecular beacon oligonucleotide is labeled with a fluorescent compound and a quencher compound, which due to the secondary structure of the molecule are in close vicinity to each other. Upon binding to the target DNA, the intramolecular hydrogen bonding is broken, and the fluorescent compound attached at one end of the probe is separated from the quencher compound, which is attached at the opposite end of the probe (Lizardi et al., U.S. Pat. No. 5, 118,801).
However, prior to the time the invention was made, there was no homogenous detection format available which allowed for the quantification of the number of sequence repeats in repetitive sequences. Nevertheless, such an analysis of repeat numbers is of major importance for example in the field of Microsatellite analysis. Microsatellites (MIS) are short tandem repeats which are distributed over the entire human genome. Micro-satellites occur statistically about once every 100,000 base pairs. Up to now 5 classes of MIS have been described which differ from one another in the length of their smallest repetitive unit as a mono-, di-, tri-, tetra- or pentanucleotide repeat. As a rule these repetitive units occur repeatedly 10 to 40 times in a tandem arrangement. Microsatellite instability (MIN) in the form of small deletions or insertions can be detected in many tumour patients if one compares DNA from tumour material with normal DNA of the same individual (Thibodeau et al., (1993), Science, 260, 816-819) (WO 94/19492). This is achieved by amplifying the DNA with the aid of PCR and subsequently separating the amplification products by gel electrophoresis. A permanent replication defect of the tumour cells is regarded to be the cause of MIN (Parsons et al., (1993), Cell, 75, 1227-1236; Shibata et al., (1994) Nat. Genet. 6, 273-281). Such tumours are classified as xe2x80x9creplication error-positivexe2x80x9d (RER+). An RER+phenotype is characteristic for colorectal tumours in families with HNPCC (hereditary non-polyposis colon cancer) (Aaltonen et al., (1993), Science, 260, 812-816).
The analysis of microsatellites is an extremely attractive method for diagnostic applications as well as for the examination of the tumourigenesis of RER+tumours. Because it is simple to carry out the determination of MIN before sequencing the mismatch repair gene of HNPCC families is a suitable aid in identifying potential RER+patients. MIN analysis is also of major importance for the prognostic diagnosis of sporadic colorectal carcinoma because the occurrence of MIN correlates with a better prognosis (Lothe et al. (1993) Cancer Res., 53, 5849-5852; Thibodeau et al. (1993), Science, 260, 816-819; Bubb et al. (1996) Oncogene, 12, 2641-2649).
MIN can be detected in more than 90% of all HNPCC tumours (Liu et al., (1996) Nature Med., 2, 169-174) whereas MIN only occurs with a frequency of 10-20% in sporadic colorectal tumours (Thibodeau et al. (1993) Science, 260, 816-819; Ionov et al. (1993), Nature, 363, 558-561; Aaltonen et al. (1993) Science, 260, 812-816; Lothe et al. (1993) Cancer Res., 53, 5849-5852). However, MIN is not restricted to colorectal tumours but has also been detected in other tumours. These include among others pancreatic carcinomas
(Han et al. (1993) Cancer Res., 53, 5087 -5089), gastric carcinomas (Han et al. (1993) Cancer Res., 53, 5087-5089; Peltomaki et al. (1993) Cancer Res., 53, 5853-5855; Mironov et al. (1994) Cancer Res., 54, 41-44; Rhyu et al. (1994) Oncogene, 9, 29-32; Chong et al. (1994) Cancer Res., 54, 4595-4597), prostate carcinomas (Gao et al. (1994) Oncogene, 9, 2999-3003), carcinomas of the endometrium (Risinger et al. (1993) Cancer Res., 53, 5100-5103; Peltomaki et al. (1993) Cancer Res., 53, 5853-5855) and mammacarcinomas (Patel et al. (1994) Oncogene, 9, 3695-3700).
Prior to the invention, analysis of Microsatellites required in any case a time consuming fragment separation by gel electrophoresis in order to detect the size of the amplification products thereby gaining information about the length of the repeat structure. Thus, there was a need in the art to provide a method for the analysis of the length of repetitive structures without fragment separation. Such a method requires to be quick and easy to perform. In addition, it is advantageous, if such a method can in principle become automated.
The new invention relates to a method, wherein the number of repeat sequences which are present in a sample is detemined by means of melting temperature analysis.
Therefore, in a first aspect, the invention relates to a method for analysis of a target nucleic acid consisting of repetitive and non repetitive sequences comprising:
a) hybridization of at least one polynucleotide hybridzation probe comprising a first segment which is complementary to a non repititive region and a second segment which is complementary to an adjacent repetitive region, said second segment consisting of a defined number of repeats
b) determination of the melting point temperature of the hybrid which has been formed between the target nucleic acid and the at least one hybridization probe.
According to the invention, the melting point temperature can then be correlated with the number of repeats present in the target nucleic acid
Usually, it is advantageous to compare the determined melting temperature value with the melting temperature value obtained for a reference nucleic acid. Therefore, the invention relates also to a method for analysis of a target nucleic acid in a sample said target nucleic acid consisting of repetitive and non repetitive sequences comprising:
a) hybridization of at least one polynucleotide hybridzation probe comprising a first segment which is complementary to a non repetitive region and a second segment which is complementary to an adjacent repetitive region, said second segment consisting of a defined number of repeats.
b) hybridization of the same polynucleotide hybridzation probe as in step a) with a target nucleic acid in a reference sample
c) determination of the melting point temperature of the hybrids which have been formed between the target nucleic acid and the at least one hybridization probe in both the sample and the reference sample
d) determination of the difference between the two melting point temperatures as a measure for the difference in repeat numbers between the target nucleic acids in the sample and the reference sample.
In order to gain increased sensitivity it has also been proven to be advantageous, if the target nucleic acid is amplified prior to hybridization. Amplification can easily be achieved for example by a Polymerase Chain Reaction.
Usually, the at least one hybridization probe is labeled and the label is preferably a fluorophore. In an even more preferred embodiment, detection according to the FRET/Hybprobe principle is performed. In this case, hybridization is performed with two adjacently hybridizing probes each labeled with a different fluorophor, such that Flourescence Resonance Energy Transfer can take place, when both probes are hybridized to the target nucleic acid.
The new method is applicable for a variety of different experimental set ups, wherein the number of repeat structures in a repetitive sequence needs to be determined.
Consequently, a specific aspect of the invention relates to an application of the new method for the analysis of Microsatellites, and especially Microsatellite Instability (MIN). Such an analysis is well applicable in order to detect hereditary tumors, especially colorectal tumors which are caused by defects in the HNPCC mismatch repair genes.
Similarily, another aspect of the invention focuses on the use of the new method for the identification of an individual in a population, for example in order to resolve forensic problems.
In a further aspect, the invention also relates to respective polynucleotide hybridization probes, comprising a first segment which is complementary to a non repititive region and a second segment which is compementary to an adjacent repetitive region, said second part consisting of a defined number of repeats. In a specific embodiment of the invention, the number of repeats present in the probe is identical to the number of repeats in the wild type of the target sequence. If there is no specific wild type, the number of repeats within the probe is preferably identical to the maximum number of repeats occuring in the repeat locus to be investigated.
Furthermore, it has been proven to be highly advantageous, in case the non repetitive segment of such hybridization probes has a length of 3-10 nucleotides, even more preferably 3, 4, 5 or 6 nucleotides.
In another aspect, the invention relates to a pair of FRET hybridization probes, wherein the first probe consists of non repetitive sequences and the second probe comprises a non repetitive region and a second segment which is complementary to an adjacent repetitive region, said second part consisting of a defined number of repeats. Preferably, in this case the label of the second probe is attached at the non repetitive region of the probe. In a still further aspect, the invention relates to specific polynucleotide hybridization probes having a sequence according to SEQ. ID. NO: 4 for analysis of BAT 26 and SEQ. ID. NO: 7 for analysis of BAT 25, which all are in accordance with the probe characteristics disclosed above.
The following examples, references, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.